1. Field of the Invention
The present invention relates generally to the fields of virology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with modified fibers for the purpose of cell-specific targeting with the additional advantages of the concomitant elimination of endogenous tropism.
2. Description of the Related Art
Recombinant adenovirus vectors are used in a number of gene therapy applications (21, 35, 38). This fact has derived principally from the high levels of gene transfer achievable with this vector approach both in vitro and in vivo. Recombinant adenovirus vectors are distinguished from other available systems by their unique ability to accomplish in situ gene delivery to differentiated target cells in a variety of organ contexts (5, 6, 9, 10, 12, 20, 25, 27, 29, 31).
One disadvantage to the use of recombinant adenoviruses for gene therapy is related to the virus' reliance on the presence of the coxsackievirus and adenovirus receptor (CAR) to achieve high levels of gene transfer. In certain settings, this may result in sequestration of recombinant virions by nontarget, yet high CAR-expressing cells, whereas the true target cells, if low in CAR, are poorly transduced. In order to compensate for this sequestration, significant escalation in the dose of administered vector is needed, increasing the risk of inducing both direct toxicity and immune responses against the vector and further compromising the overall efficacy of the therapy. Therefore, the utility of the present generation of adenovirus vectors for gene therapy may be significantly improved by achieving targeted transduction of specific cell types by the virus.
Despite this property, specific aspects of the adenovirus biology have prevented the full realization of the potential of such vectors. In this regard, the broad tropism profile of the parent virus for cells of diverse tissues potentially allows unrestricted gene delivery. Thus, for the many gene therapy applications requiring targeted, cell-specific gene delivery, the promiscuous tropism of the adenovirus vector represents a confounding factor. Based on this concept, strategies to modify the native tropism of adenovirus have been developed to allow the derivation of vectors capable of targeted gene delivery.
Strategies to achieve this end are directed at modifying specific steps in the adenoviral infection pathway. Adenoviruses of serotypes 2 and 5 normally achieve initial recognition and binding to target cells by means of interactions between the carboxy-terminal knob domain of the fiber protein and the primary receptor (4, 17, 36). After binding, RGD motifs in the penton base interact with cellular integrins of the αvβ3 and αvβ5 types (1-3, 37, 39, 40). This interaction triggers cellular internalization whereby the virions achieve localization within the endosome. Acidification of the endosome elicits conformational changes in capsid proteins, allowing their interaction with the endosome membrane in a manner that achieves vesicle disruption and particle escape.
Following endosomolysis, the virion translocates to the nucleus, where the subsequent steps of the viral life cycle occur. This understanding of the key role played by capsid proteins in the viral infectious pathway has suggested strategies to alter this process via modifications of these proteins.
In this regard, genetic retargeting of adenovirus vectors via modification of viral genes encoding coat proteins, if successful, offers a simple way to achieve a significant improvement in the present generation of these gene delivery vehicles. To this end, several groups have reported genetic modifications to the knob domain of adenovirus fiber protein and incorporation of such chimeric fibers into virion. For instance, Stevenson et al. and others reported successful generation of AdS virions containing fibers consisting of the tail and shaft domains of Ad5 fiber and the knob domain of Ad3, respectively. In addition, Michael et al. demonstrated the incorporation of the gastrin-releasing peptide into the carboxy terminus of recombinant Ad5 fiber. This finding was extended by Legrand et al. who achieved rescue of recombinant adenovirus vectors containing such fibers. Wickham et al. described the generation of recombinant virus containing fibers with carboxy-terminal polylysine sequences. These studies have established key feasibility issues with respect to this genetic approach but have also demonstrated a number of limiting factors.
All of these modifications of adenovirus fiber were directed towards the carboxy terminus of the protein. In addition, these efforts were initiated without prior knowledge of the three-dimensional (3D) structure of the fiber knob. Thus, the employment of the carboxy terminus of the fiber represented a choice not fully incorporating all relevant considerations. Clearly, 3D structural information has important bearing upon the placement of heterologous protein sequences within the knob for targeting purposes. Such localization of targeting ligands would ideally be achieved in a manner to allow their surface presentation and to minimally perturb the fiber quaternary structure.
To overcome the limitations imposed by the CAR-dependence of adenovirus infection, the incorporation of small peptide motifs possessing receptor binding specificities into the carboxy terminal of adenovirus fiber protein has been proposed, thus enabling the virus to attach and infect via a novel cell surface receptor. This concept has been developed by Wickham et al., who have proven the feasibility of this approach by generating several recombinant adenoviruses containing fibers with targeting ligands positioned at the carboxy terminal of the fiber molecule.
Although in some cases, genetic modification of the carboxy terminal of adenovirus fiber has proven its utility with respect to vector retargeting, it has failed in some others, suggesting that this locale in the fiber molecule is not an optimal site for incorporation of targeting protein moieties. Published findings strongly suggest that addition of more than 25-30 amino acid residues of heterologous protein sequence to the carboxy terminal of the fiber molecule has dramatic negative effect on the stability of the fiber trimer and, therefore, is incompatible with the fiber functions. In addition, the three-dimensional structure of the fiber knob indicates that the carboxy terminal of the fiber points towards the virion, that is, away from the cell surface, thereby providing suboptimal environment for the incorporation of targeting ligands.
The prior art is deficient in the lack of effective means of incorporating heterologous protein sequences into the fiber knob protein of adenovirus for purposes of retargeting. The present invention fulfills this longstanding need and desire in the art.